Our goal has been to define the important molecular events that control T cell maturation within the thymus. Within the thymus, hematopoietic precursor cells undergo a complex set of developmental events and emerge as mature T lymphocytes capable of specific antigen recognition. This developmental process appears to result from a series of signalling interactions between T cell precursors and a heterogeneous and as yet ill-defined set of stromal cells. In addition to T cells, a class of cytotoxic lymphocytes known as "natural killer" cells, which play potentially important roles in immune responses against cancerous or virally-infected cells, can also develop in the thymus. We are taking two approaches to thymic development at the molecular level. First, we have established a culture system in which day 14/15 triple negative fetal thymocytes can be stimulated to express early markers of thymocyte development including CD25, ICAM-1, and Ly-6A/E. We are studying the molecular signals that activate this early developmental step. During these studies we have discovered a novel precursor cell that gives rise to both T lymphocytes and natural killer cells. We have also obtained evidence for a novel developmental pathway that allows triple negative thymocytes to develop into double positive thymocytes. Second, we are creating genetically-engineered mice that are homozygous-deficient (knocked-out) for various genes that are highly expressed in thymocytes. We are currently focusing on three genes: i) Ly-GDI - a GDP-dissociation inhibitor protein that controls the activity of Rho, a member of the Ras family that may be involved in molecular signalling cascades; ii) Rlk - a newly described member of the btk/itk family of tyrosine kinases that have been recently implicated in inherited immunodeficiency states and are involved in signalling from surface antigen receptors; and iii) Ntk - a newly-described tyrosine kinase related to the Csk kinase that regulates various Src family kinases by phosphorylating inhibitory tyrosine residues. Since signalling interactions are critical for proper T cell differentiation, disruption of these signalling molecules may inhibit T cell development in ways that clarify the normal physiological roles of these molecules.